Melatonin-induced methylation of the ABCG2/BCRP promoter as a novel mechanism to overcome multidrug resistance in brain tumour stem cells.

BACKGROUND: Current evidence indicates that a stem cell-like sub-population within malignant glioblastomas, that overexpress members of the adenosine triphosphate-binding cassette (ABC) family transporters, is responsible for multidrug resistance and tumour relapse. Eradication of the brain tumour stem cell (BTSC) compartment is therefore essential to achieve a stable and long-lasting remission. METHODS: Melatonin actions were analysed by viability cell assays, flow cytometry, quantitative PCR for mRNA expression, western blot for protein expression and quantitative and qualitative promoter methylation methods. RESULTS: Combinations of melatonin and chemotherapeutic drugs (including temozolomide, current treatment for malignant gliomas) have a synergistic toxic effect on BTSCs and A172 malignant glioma cells. This effect is correlated with a downregulation of the expression and function of the ABC transporter ABCG2/BCRP. Melatonin increased the methylation levels of the ABCG2/BCRP promoter and the effects on ABCG2/BCRP expression and function were prevented by preincubation with a DNA methyltransferase inhibitor. CONCLUSION: Our results point out a possible relationship between the downregulation of ABCG2/BCRP function and the synergistic toxic effect of melatonin and chemotherapeutic drugs. Melatonin could be a promising candidate to overcome multidrug resistance in the treatment of glioblastomas, and thus improve the efficiency of current therapies.

Fas/Fas ligand regulation mediates cell death in human Ewing's sarcoma cells treated with melatonin.

BACKGROUND: Despite recent advances in cancer therapy, the 5-year survival rate for Ewing's sarcoma is still very low, and new therapeutic approaches are necessary. It was found previously that melatonin induces cell death in the Ewing's sarcoma cell line, SK-N-MC, by activating the extrinsic apoptotic pathway. METHODS: Melatonin actions were analysed by metabolic viability/survival cell assays, flow cytometry, quantitative PCR for mRNA expression, western blot for protein activation/expression and electrophoretic mobility shift assay for transcription factor activation. RESULTS: Melatonin increases the expression of Fas and its ligand Fas L, this increase being responsible for cell death induced by the indolamine. Melatonin also produces a transient increase in intracellular oxidants and activation of the redox-regulated transcription factor Nuclear factor-kappaB. Inhibition of such activation prevents cell death and Fas/Fas L upregulation. Cytotoxic effect and Fas/Fas L regulation occur in all Ewing's cell lines studied, and do not occur in the other tumour cell lines studied where melatonin does not induce cell death. CONCLUSION: Our data offers new insights in the study of alternative therapeutic strategies in the treatment of Ewing's sarcoma. Further attention deserves to be given to the differences in the cellular biology of sensitive tumours that could explain the cytotoxic effect of melatonin and the increase in the level of free radicals caused by this molecule, in particular cancer types.

Antioxidants do not prevent acrylonitrile-induced toxicity.

Several reports have recently described that acrylonitrile (ACN) toxicity resides in its capacity for inducing oxidative stress. ACN can be conjugated with glutathione (GSH), diminishing its cellular content, or being metabolized to cyanide. In the present report, we determine the effect of ACN on the viability of primary-cultured astrocytes as well as the oxidative damage generated by ACN by measuring GSH levels in primary cultured astrocytes. We also analyzed whether the ACN (2.5mM) toxicity could be avoided by using antioxidants such as taurine (5mM), N-acetylcysteine (20 mM), trolox (100 microM), estradiol (10 microM) and melatonin (100 nM-1mM). In this cell culture model, antioxidants were not able to prevent ACN-induced cell damage, with the exception of NAC, confirming that only GSH seems to play a key role in ACN-derived toxicity. Additionally, we measured different parameters of oxidative stress such as catalase activity, lipid peroxidation and GSH concentration, as indicators of the potential oxidative stress mediated by the toxicity of ACN, after exposure of Wistar rats to a concentration of 200 ppm ACN for 14 days. At the concentration assayed, we did not find any evidence of oxidative damage in the brain of ACN-treated rats.

Androgen regulation of gene expression in the Syrian hamster Harderian gland.

The androgenic control of sexual dimorphism has been studied in the Harderian gland from Syrian hamster and compared to rat Harderian gland, a system without dimorphism. Hybridization in situ with a rat cDNA clone has revealed the presence of androgen receptor mRNA in all secretory cells from male and female hamster glands. Testosterone or 5-alpha-dihydrotestosterone administration to females both caused a 60% decrease in the levels of 5-aminolevulinate synthase mRNA after 1 day of treatment, but the resulting patterns of in vitro translation using RNA from glands treated with the two androgens are different. Testosterone alters the mRNA levels for androgen receptor and 5-aminolevulinate synthase in the glands only 6 h after its implantation in females, and the action is maintained up to 10 days of treatment. Finally, androgen administration to females or deprivation in males alter androgen receptor but not 5-aminolevulinate synthase mRNA levels in rat Harderian glands. Our results suggest that the androgen receptor from Harderian glands is responsible for the sexual dimorphism found in Syrian hamsters, whereas the lack of sexual dimorphism in rat seems to be due to a restricted effect of androgens in the glands.

Mast cells in the Harderian gland of female Syrian hamsters during the estrous cycle and pregnancy: effects of the light/dark cycle.

The number of identifiable mast cells and the intraluminal area occupied by porphyrin deposits was studied on semithin sections from female hamster Harderian glands during the estrous cycle and pregnancy. Although the serum levels of estradiol, progesterone, luteinizing hormone and follicle stimulating hormone exhibited significant changes throughout the cycle, no correlation between these changes and the variations in the number of recognizable mast cells was observed. However both during diestrous 1 and proestrous cycles, the number of identifiable mast cells was higher at midnight than at noon (in 14 h light:10 h dark photoperiod with lights on at 07:00 h). A more exhaustive study revealed the presence of 'degranulated mast cells' which were not stained with toluidine blue. Thus, a diurnal cycle in degranulation might occur in the Harderian glands from female hamsters. No significant variations were observed in the area occupied by intraluminal porphyrin deposits during the estrous cycle. However, both the relative number of mast cells and the area occupied by intraluminal porphyrins decreased from day 4 of pregnancy to day 14 showing a strong correlation. The Harderian glands from female Syrian hamsters might provide a useful model for the study of mast cell degranulation during porphyria.

Isolation and identification of sex-specific cDNA clones from the Syrian hamster harderian gland.

Syrian hamster Harderian glands show a typical sexual dimorphism, with males having two secretory cell types and females having one cell type and intraluminal porphyrin accretions, among other differences. Since these differences may be due to the expression of specific genes, our interest is to identify those genes and their role on the development and control of the sexual dimorphism. The experimental approach was to construct cDNA libraries for male and female Syrian hamster Harderian glands and then subtracted libraries for male vs. female and for female vs. male. By this method, cDNA libraries enriched either in male-specific or in female-specific clones were obtained. Clones from those libraries were checked for differential expression by using double colony hybridization with [32P]-cDNA from male and female glands. Then, the selected clones were checked again for expression in Harderian glands by Northern hybridization, using poly(A+) RNA from males, castrated males, and females. Finally, the clones were sequenced and compared to search for significant homologies. One of the male-specific clones showed strong homology with rat cytochrome p450b/e. Among the female-specific clones, homologies were found to the complement C3 fragment from several species, to sequences from the mouse mammary tumor virus, and to the subunits C1 and C2 of the rat prostatic steroid binding protein. Several other clones showed no significant homologies and need further characterization.

Invasive processes in the normal Harderian gland of Syrian hamster.

In this contribution we will pay special attention to several morphological findings that we can observe, under some circumstances, in the normal Harderian gland of the Syrian hamster. The accumulation of porphyrins in this gland results in mitochondrial damage and extensive cell death. Many damaged cells are secreted into the lumen of the tubule-alveoli, but most of them seem to produce an invasive process that even affects the vascular components of the gland. In this way, many blood vessels are invaded and appear partially filled with the invasive mass, which sometimes totally occludes the lumen of the vessels. We have also observed other surprising features related to a special kind of activity in certain secretory cells. Such activity results in a peculiar "segregation" of a cytoplasmic fragment, containing the nucleus. The affected cells seem to gather up their cytoplasm and nucleus towards the basal zone, while the rest of the cell, including practically the whole amount of lipid droplets, is relegated to the vicinity of the lumen. All these phenomena seem finally to result in the detachment of some clusters, composed of a limited number of cells, which display a basophilic cytoplasm practically free of lipid droplets.

Regulation of the aminolevulinate synthase gene in the Syrian hamster Harderian gland: changes during development and circadian rhythm and role of some hormones.

The Syrian hamster Harderian gland has been advocated as a model to study the porphyrin biosynthetic pathway, since it shows by far the highest porphyrin concentration known to date. Another particular characteristic is the sexual dimorphism at both the morphological and the biochemical levels. We found a variation in the ALV-S (aminolevulinate synthase) gene expression according to sex, with females exhibiting much higher mRNA levels than do males. After castration, ALV-S mRNA rose considerably in males, this increase being inhibited by darkness or treatment with melatonin. Treatment with hCG or progesterone did not vary the ALV-S mRNA levels in females. Castrated males, however, showed a much larger increase when they were treated with hCG. No variations have been found in the expression of the ALV-S gene in female HG throughout the estrous cycle. During development, males and females showed similar ALV-S mRNA levels until they were 20 days old. Afterwards, they started showing gender-associated differences. In females, ALV-S mRNA levels rose during the first 3 months of life, and thereafter they decreased progressively with aging. A circadian rhythm has been found in the gene expression of ALV-S mRNA in females, showing very low levels in the morning and reaching a peak during the first hours of darkness. It was an endogenous rhythm, probably regulated at the transcriptional level. It is proposed that the light-dark period duration modulates this rhythm through the suprachiasmatic nucleus which in turn acts on the pineal secretion of melatonin that regulates ALV-S gene expression.

Ultrastructural confirmation of neuronal protection by melatonin against the neurotoxin 6-hydroxydopamine cell damage.

6-Hydroxydopamine (6-OHDA) is a neurotoxin used in the induction of experimental Parkinson's disease in both animals and cultured neuronal cells. Biochemical and molecular approaches showed previously that low doses of 6-OHDA induced apoptosis in PC12 cells, while high doses of this neurotoxin induced necrosis. Melatonin has been shown to protect against the neuronal programmed cell death induced by 6-OHDA, although it was not able to prevent the massive necrotic cellular death occurring after the addition of high doses of the neurotoxin. In the present work, we demonstrate by ultrastructural analysis that although low doses of 6-OHDA induced apoptosis in PC12 cells, it also damaged the non-apoptotic cells, morphologically corresponding this damage to incipient and reversible necrotic lesions. When the doses of the neurotoxin increase, there are still apoptotic cells, although most of the cells show necrotic irreversible lesions. We also found that melatonin partially prevents the incipient necrotic lesions caused by low doses of 6-OHDA. The fact that melatonin was shown in previous work to prevent apoptosis caused by low doses of 6-OHDA, but not necrosis induced by high doses of the neurotoxin, seemed to indicate that this agent is only able to protect against apoptosis. However, our present results, melatonin preventing also the incipient necrotic neuronal lesions, suggest that this hormone may provide a general protection against cell death, suggesting that higher doses should be tried in order to prevent the necrotic cell death induced by high doses of the neurotoxin.

Melatonin decreases mRNA for histone H4 in thymus of young rats.

The antiproliferative properties of melatonin have been previously demonstrated for several normal and tumoral tissues. In a recent report we have shown that melatonin is able to inhibit programmed cell death in thymus both, in vivo and in vitro. Given that other authors have related programmed cell death and cell proliferation and that no previous reports on melatonin and cell division exist on thymus, we decide to study the possible antiproliferative effect of melatonin in this organ measured as the levels of mRNA for the histone H4. We found that melatonin inhibits cell division on thymus when administered chronically both, at high (500 microg/body weight) and low (50 microg/body weight) dose. We also found a circadian rhythm of the mRNA for histone H4, opposed to the one previously described for melatonin, supporting the negative regulation by this hormone of cell division on thymus. A single dose of melatonin (50 microg/body weight) was not able to decrease the levels of mRNA for H4 in the time-points studied but after two hours of its administration. Finally, we report the inhibitory effect of melatonin in the cell proliferation of Harderian gland, brain, lung and kidney.

Development and hormonal regulation of mast cells in the Harderian gland of Syrian hamsters.

The morphological features and relative number of mast cells per mm2 were studied in the Harderian glands of male and female Syrian hamsters (Mesocricetus auratus) under different experimental conditions. The structural and ultrastructural characteristics of Harderian mast cells corresponded to those of connective tissue mast cells. The Harderian glands from female hamsters contained more mast cells than those of male hamsters. A subcutaneous implant of testosterone (2 mg/24 mg beeswax) resulted in a rapid decrease in the number of recognizable mast cells 6 h after the implantation. Neither orchidectomy nor ovariectomy significantly altered the relative number of mast cells. However, the daily subcutaneous injection of 20 IU of human chorionic gonadotropin during 20 days resulted in a significant decrease of identifiable mast cells. The administration of another steroid such as progesterone or the induction of states of hypo- and hyperthyroidism did not alter the distribution of mast cells in the Harderian glands of female Syrian hamsters.

Androgen-dependent mast cell degranulation in the Harderian gland of female Syrian hamsters: in vivo and organ culture evidence.

In previous articles we have reported the "disappearance" of Harderian gland mast cells (HGMC) after treatment with testosterone. In the present work we study: (a) if the apparent decrease in the number of mast cells caused by this androgen is real or is due to the fact that testosterone induces mast cell degranulation that avoids its recognition by toluidine blue staining; (b) if testosterone acts through its receptor directly on the Harderian gland (HG). In order to give an answer to the first question, we observed HG of female Syrian hamsters treated with testosterone under the electron microscope to find the possible degranulated mast cells not recognizable with the aid of the toluidine blue staining. We also studied in vivo and in vitro the effects of the beta-agonists isoproterenol and salbutamol, given that they increase cAMP and can therefore prevent degranulation of mast cells. Finally we have used cytocalasin B, which inhibits degranulation by blocking actin depolimerization. Both the beta-agonists and cytochalasin B were able to prevent the decrease of mast cells, as recognized by staining with toluidine blue after treatment with testosterone. Indeed, when observed under the electron microscope, abundant degranulated mast cells were found after treatment with testosterone. For solving the second issue we analyzed the effect of the antiandrogen cyproterone acetate in vivo and in vitro. Our results demonstrate that testosterone is able to induce degranulation of HGMC in the Syrian hamster Mesocricetus auratus and that this effect is achieved directly through its receptor on the Harderian gland.

Antioxidative protection in a high-melatonin organism: the dinoflagellate Gonyaulax polyedra is rescued from lethal oxidative stress by strongly elevated, but physiologically possible concentrations of melatonin.

Lethal oxidative stress was investigated in the dinoflagellate Gonyaulax polyedra by measuring the dying-peak of bioluminescence during circadian phases of low physiological light emission, low bioluminescence capacity, and low sensitivity to stimulatory agents. Measurements were carried out in constant darkness after transfer of cells from light at CT 6 (circadian time, 0600 hr). H2O2 (0.08 mM), when administered 1 hr after transfer of cells, led to a multifold, long-lasting enhancement of light emission, which is typical for lethal cell damage. At the circadian phases of investigation, melatonin did not substantially stimulate bioluminescence up to concentrations of 0.5 mM. At this concentration, addition of melatonin prevented the dying-peak and reduced bioluminescence to almost basal values. The high concentration of melatonin applied is not unphysiological in Gonyaulax, because the indoleamine can increase to levels of several millimolar, e.g., in response to temperature signals. These protective effects of melatonin seem to be caused mainly by the direct action of melatonin as an antioxidant, because the major enzymes of antioxidative protection were not stimulated by melatonin, although some of them responded to H2O2. The activities of neither superoxide dismutase, hemoperoxidase/catalase, glutathione peroxidase, nor haloperoxidase were enhanced under the influence of melatonin; glutathione S-transferase activity increased only slightly.

The pineal gland of the trumpet-tailed rat (Octodon degus).

The structure and ultrastructure of the pineal gland of the degu or trumpet-tailed rat (Octodon degus), a rodent inhabiting tropical-equatorial areas, was examined under light and electron microscopy. On the basis of its form, size, and location, the pineal gland of the degu is classified as a proximal or "A" type. The connective tissue appeared poorly developed and the gland contained non-fenestrated capillaries. A single population of typical pinealocytes was found. In addition, a small number of glial cells and cells with electron dense bodies appeared scattered throughout the gland. Cells with dense granules were found isolated or forming small groups always in close proximity to blood vessels. Numerous sympathetic nerve fibers with small dense-core vesicles were found. Also, some myelinated nerve fibers were observed. The physiological significance of the presence of large electron-dense granules in some pineal cells and their particular location around the blood vessels in discussed.

Androgenic control of porphyrin in the Harderian glands of the male Syrian hamster is modulated by the photoperiod, which suggests that the sexual differences in porphyrin concentrations in this gland are important functionally.

BACKGROUND: The porphyrin concentrations of the Harderian glands of Syrian hamsters show marked sexual differences, with male levels being much lower than those of females. Porphyrinogenesis is inhibited by androgens, so orchidectomy leads to elevated male porphyrin concentrations; however, a number of other procedures (some of which also lower androgen levels) prevents this. We studied the effects of short-day photoperiods and melatonin on Harderian porphyrin concentrations. METHODS: Intact, castrated, or pinealectomized hamsters of both sexes were exposed to long-day or short-day photoperiods. Intact or castrated hamsters were given melatonin injections in the morning or the afternoon, or were given beeswax pellets containing melatonin. After a variable period, Harderian glands were dissected and porphyrins were measured. RESULTS: Prolonged short-day exposure (13 weeks) led to increased Harderian porphyrin concentrations and this rise was prevented by pinealectomy. The rise in Harderian porphyrins following short-day exposure was small, compared with that following castration. Short-day photoperiods also prevented the rise in porphyrin levels associated with castration and this effect was prevented by removal of the pineal. Melatonin injections, whether given in the morning or in the afternoon, had no effect on Harderian porphyrin concentration of castrated male hamsters. Continuous release melatonin pellets reduced the postcastrational rise in porphyrin levels in one experiment, while having no effect in another. In female hamsters, neither short photoperiods nor melatonin pellets influenced Harderian porphyrin concentrations. CONCLUSIONS: These results suggested that a factor from the pineal gland helps maintain the low levels of porphyrin which are characteristic of male Harderian glands, despite the decrease in androgen levels which typically results from exposure to short days. Morning and afternoon injections of melatonin and continuous release melatonin pellets failed to resolve the question of whether this pineal factor is melatonin. Our results demonstrated that low male and high female porphyrin levels are maintained in Syrian hamsters, despite seasonal variations in the hormonal milieu, suggesting that these sexual differences are important for the (still unestablished) function of the Harderian glands in this species.

Lymphoid cells in the harderian gland of the rodent Octodon degus.

The Harderian gland of the degu (Octodon degus) is composed of tubulo-alveolar secretory units that share most of morphological features found in the Harderian glands of other rodents. However, a peculiar characteristic observed in the glands of female degus is the existence of lymphoid cell clusters within the connective tissue surrounding the secretory adenomeres. Lymphocytes and lymphoblasts are found associated with blood vessels and especially with nerve bundles in the medullary region of the gland. Occasionally, macrophages and plasma cells are also observed. Although the Golgi apparatus appears well developed, the ultrastructural characteristics of most of these lymphoid elements correspond to those of inactive lymphocytes. Unmyelinated fibers containing clear and dense-core vesicles are found closely related to lymphocytes. On some occasions, lymphocytes present extensive areas of apposition with structures resembling intercellular junctions. The analogy of the lymphoid clusters reported in this study with those described in the avian Harderian gland is discussed.

Melatonin increases gene expression for antioxidant enzymes in rat brain cortex.

During the last years several reports have demonstrated that melatonin is a efficient free radical scavenger and general antioxidant. In addition, it has been shown that this neurohormone is able to increase the activity of glutathione peroxidase in rat brain cortex as well as the gene expression for some antioxidant enzymes in the Harderian gland of female Syrian hamster. Also, it is well known that brain cells are particularly exposed to free radicals, with antioxidant enzymes as the major defense mechanism that the brain uses to neutralize reactive oxygen species. The aim of the present study was to examine the influence of melatonin on gene expression for antioxidant enzymes in rat brain cortex. Our results clearly demonstrate that exogenously administered melatonin increases the levels of mRNA for glutathione peroxidase, copper-zinc superoxide dismutase, and manganese superoxide dismutase in this tissue. These stimulatory effects are observed after both acute and chronic treatment with this hormone, producing in the latter case the more marked increase. We therefore conclude that melatonin exerts an important role in providing indirect protection against free radical injury by stimulating gene expression for antioxidant enzymes. Consequently, melatonin could be considered as a potential therapeutic agent in some age-related neurodegenerative diseases where excessive free radical production has been implicated.

Glutamate induces oxidative stress not mediated by glutamate receptors or cystine transporters: protective effect of melatonin and other antioxidants.

Glutamate is responsible for most of the excitatory synaptic activity and oxidative stress induction in the mammalian brain. This amino acid is increased in the substantia nigra in parkinsonism due to the lack of dopamine restraint to the subthalamic nucleus. Parkinson's disease also shows an increase of iron levels in the substantia nigra and a decrease of glutathione, the antioxidant responsible for the ascorbate radical recycling. Considered together, these facts could make the antioxidant ascorbate behave as a pro-oxidant in parkinsonism. Since both glutamate and ascorbate are present in the synaptosomes and neurons of substantia nigra, we tested 1) if glutamate is able to induce oxidative stress independently of its excitatory activity, and 2) if ascorbate may have synergistic effects with glutamate when these two molecules co-exist. Brains were homogenized in order to disrupt membranes and render membrane receptors and intracellular signaling pathways non-functional. In these homogenates glutamate induced lipid peroxidation, indicating that this amino acid also may cause oxidative stress not mediated by its binding to glutamate receptors or cystine transporters. Ascorbate also induced lipid peroxidation thus behaving as a pro-oxidant. Both substances together produced an additive effect but they did not synergize. Given that melatonin is a potent physiological antioxidant with protective effects in models of neurotoxicity, we tested the role of this secretory product on the pro-oxidant effect of both compounds given separately or in combination. We also checked the protective ability of several other antioxidants. Pharmacological doses of melatonin (millimolar), estrogens, pinoline and trolox (micromolar) prevented the oxidant effect of glutamate, ascorbate, and the combination of both substances. Potential therapeutic application of these results is discussed.

Inhibition of cell proliferation: a mechanism likely to mediate the prevention of neuronal cell death by melatonin.

In a previous work we demonstrated that melatonin is able to prevent apoptosis induced by low doses of 6-hydroxydopamine (6-OHDA) in undifferentiated and neuronal PC12 cells. We also reported how this neurohormone was able to prevent the decrease in the mRNA for antioxidant enzymes caused by 6-OHDA. Although the antioxidant capability of melatonin seems to be clearly implicated in its antiapoptotic activity, literature suggests that its antiproliferative property could also be involved in its prevention of apoptosis. In the present work we demonstrated that melatonin is able to inhibit cell proliferation in undifferentiated PC12 cells, decreasing cell number and the total amount of DNA, and the mRNA for the histone H4, which are known to increase during DNA synthesis. Melatonin does not decrease the number of cells in nonproliferating PC12 cells, indicating that it does not cause cell death. Additionally, we demonstrate that other inhibitors of cell proliferation, as well as other antioxidants, are able to mimic the antiapoptotic effect of melatonin. This is interpreted to mean that melatonin acts by both mechanisms to inhibit apoptosis caused by 6-OHDA and the findings support the hypothesis of a relationship between oxidative stress and regulation of the cell cycle.